Axon-TRAP-RiboTag: Affinity Purification of Translated mRNAs from Neuronal Axons in Mouse In Vivo.

TitleAxon-TRAP-RiboTag: Affinity Purification of Translated mRNAs from Neuronal Axons in Mouse In Vivo.
Publication TypeJournal Article
Year of Publication2018
AuthorsShigeoka, T, Jung, J, Holt, CE, Jung, H
JournalMethods Mol Biol
Volume1649
Pagination85-94
Date Published2018
ISSN1940-6029
KeywordsAnimals, Axons, Breeding, Chromatography, Affinity, Immunoprecipitation, Mice, Protein Biosynthesis, Ribosomes, RNA
Abstract

Translating ribosome affinity purification (TRAP) is a widely used technique to analyze ribosome-bound mRNAs in particular target cells that express a tagged ribosomal protein. We developed axon-TRAP-RiboTag, a TRAP-based method that allows purification and identification of translated mRNAs from distal neuronal axons in mouse, and identified more than 2000 of translated mRNAs in retinal ganglion cell (RGC) axons in vivo. The use of Cre-negative littermate control to filter out false-positive signals allows unbiased detection, and combining TRAP with in vitro ribosome run-off enables identification of actively translated mRNAs. Here, we describe a detailed protocol to identify translated mRNAs in RGC axons in mouse in vivo. This method can be applied to any neurons whose cell bodies and distal axons are anatomically separated.

DOI10.1007/978-1-4939-7213-5_5
Alternate JournalMethods Mol. Biol.
Citation Key10.1007/978-1-4939-7213-5_5
PubMed ID29130191
Grant List085314/Z/08/Z / / Wellcome Trust / United Kingdom